A little more than 2 decades ago, screening for
autoantibodies associated with Type 1A (immune mediated) diabetes was limited
to measuring cytoplasmic islet cell autoantibodies (ICAs) and insulin
antibodies 1, 2.
Currently, multiple sequenced autoantigens have been defined and recombinant
autoantibody assays3-7
are available 8, 9including assays for insulin10,
glutamic acid decarboxylase (GAD) 11,
ICA512/IA-2 12-14, I-A2 beta (phogrin) 15, 16, and the islet zinc
transporter, ZnT8 autoantibodies 17, 18.Only autoantibody assays to these proteins
are of confirmed utility in diagnosing and predicting Type 1A (autoimmune
diabetes) and for which laboratories participating in blinded workshops
organized by the Immunology of Diabetes Society submit measurements.A long list of additional autoantigens with
autoantibody assays exist (see below) but in general we believe given lack of
implementation one should assume that these other proposed assays lack
sensitivity and or specificity, and the proposed targeted antigen is not
sufficiently Type 1A diabetes associated to be of diagnostic utility.In addition to identifying the appropriate
target molecules as the basis for Type 1A diabetes autoantibody assays, the
assay format is also crucial.Fluid
phase radioassays have historically provided the best sensitivity and
specificity but this may be changing.Standard ELISA assays where one simply binds a given autoantigen to a
plate and detects binding of autoantibodies to the plate bound antigen in
general have lacked requisite specificity when large numbers of samples are
analyzed.A modified capture ELISA
format where autoantibody cross-links a plate bound antigen to the same labeled
fluid phase antigen for GAD65, IA-2, and ZnT8 have approached the specificity
and sensitivity of fluid phase radioassays.In addition two additional formats (luciferase based assays19
and electrochemiluminescent20)
are being studied.The
electrochemiluminescent assay format has been applied to the insulin
autoantibody which is the most difficult for laboratories to implement 20, 21
with a marked increase in sensitivity above that of even radioassays as
evaluated in the last two international Immunology of Diabetes workshops (DASP
and IASP).

Just amongst the four major islet autoantibody assays,
insulin, GAD65, IA-2, and ZnT8 there are major differences.Insulin autoantibodies are almost always the
first autoantibody to appear in children followed from birth (either as an
isolated autoantibody or with other autoantibodies)22.The levels of insulin autoantibodies (and
only insulin autoantibodies) correlate with the rate of progression to onset of
overt diabetes from the time of first appearance of an islet autoantibody6, 23.Thus the youngest onset children who must
progress rapidly to be onset at a young age, at onset characteristically have
very high levels of insulin autoantibodies.The levels of insulin autoantibodies are not simply related to age, as
in general the levels are very low for the whole prediabetic period in young
autoantibody positive children who take a long time (e.g. decade) to progress
to diabetes(Figure 10.1 below).A recent
report suggests that the prevalence and levels of GAD and IA-2 autoantibodies
at diabetes onset have increased between 1985 and 200224.

Figure 10.1.Child followed from birth in DAISY study till
development of overt diabetes.Autoantibodies were present prior to two years of age with more than a
dozen years till onset of hyperglycemia.Characteristically for children who slowly progress to overt diabetes is
the lack or low levels of insulin autoantibodies.

The most specific islet autoantibodies react with IA-2 and
ZnT8 18, 25
and for these two autoantibodies there is characteristically a very strong
signal to noise.GAD65 autoantibodies
are the most common amongst adult onset patients, but GAD65 autoantibody assays
frequently have a 3-5% positivity rate in controls 26
making definitive diagnosis of Type 1A diabetes more difficult if GAD is the
only autoantibody present (e.g. diagnosis of Latent Autoimmune Diabetes of
Adults) if only GAD65 autoantibodies are present.Though insulin autoantibodies are present in
almost all children followed to diabetes, they are frequently negative at onset
of diabetes in teenagers and adults.

Expression of multiple autoantibodies of insulin, GAD, IA-2
and/or ZnT8 is associated with extreme risk of progressing to overt Type 1A
(immune mediated) diabetes25, 27, 28.Given 20 years of follow up almost all
children (whether relatives of patients with Type 1 diabetes or individuals
from the general population) who express multiple biochemical autoantibodies
progress to diabetes.If there is only a
single autoantibody, which are usually insulin autoantibodies or GAD
autoantibodies risk of diabetes is low, often less than 5%25, 29.As the number of autoantibodies increase the
overall risk of diabetes increases29.The usual explanation for the general rule
relating multiple autoantibodies to extreme risk is that epitope spreading with
the targeting of different autoantigens directly enhances beta cell
destruction.Though insulin
autoantibodies often appear first in prediabetic children followed from birth,
the order of autoantibody appearance is very variable.After initial appearance with either multiple
or a single autoantibody relatively rapidly multiple autoantibodies are present
which persist till the development of diabetes.It is doubtful that there is one to one correlation between
autoantibodies and T cell targeting of specific islet antigens.In particular with only the level of insulin
autoantibodies related to the rate of progression, subjects with low or
negative insulin autoantibodies,individuals may still be critically targeting insulin at a low level
consistent with delayed progression.Rate of the process needs to be distinguished from risk and it is
easiest to do this by only analyzing individuals who have all progressed to
overt diabetes with prospective follow up.A life table mixing individual with and without progression to diabetes
reflects both rate and risk23.

An alternative hypothesis for the >=2 autoantibody rule,
given that Type 1 diabetes related autoimmunity is relatively rare (e.g. 1/300
in general population developing diabetes) is statistical. By Baye’s theorem if
one is measuring four autoantibodies (each assay with 1% false positive rate
[which only the best international assays achieve in Immunology of Diabetes
Society workshops]) the great majority of positives in general population
screening are likely to be “false” positives in terms of development of
diabetes.A simple way to increase
testing specificity is to utilize multiple biochemical autoantibody assays with
definition of positive as the presence of >=2 of the four autoantibodies
(binomial equations).The combinatorial
approach rather than simply asking for all four autoantibodies to be present
(four autoantibodies present predicted specificity =(.01)4 or
>99.99999%) allows one to preserve sensitivity with a decreased but
impressive specificity27.False positives in the general sense for
islet autoantibody results can result from assay problems (e.g. we have
observed an individual with autoantibodies reacting only with iodinated insulin
which does not exist in nature but I-125 iodinated insulin is utilized for the
insulin autoantibody radioassay).False
positives can also result from true autoantibodies which have low positive
predictive values (e.g. “biologic” false positives, low affinity insulin
autoantibodies20, 22,
ELISA assays for insulin autoantibodies not detecting diabetes related
autoantibodies10, etc.).Finally false positives may result from
non-antibody serum components that bind or precipitate labeled
autoantigens.

Once an individual is identified with islet autoantibodies,
follow up for deterioration of glycemic control is important as it is clear
from multiple prospective studies that ketoacidosis with both its attendant
morbidity and mortality is preventable30
especially in children less than age 2.

In addition to the highlighted major islet autoantigens,
there are many other autoantigens in a variety of stages of characterization,
including molecules with characterized sequences but without fully developed
assays and proteins with only known molecular weights of 155 kd 31, 32,
52 kd 33,
and molecules recognized by T lymphocytes for which autoantibodies have not
been described such as chromagranin Aand IGRP 34, 35 and RegII 36. Other molecules have
been described but their association with Type 1A diabetes has either not been
evaluated in man, has not been substantiated, or assay sensitivities are low,
or follow-up studies have not been published. These molecules include
carboxypeptidase H,anti-bovine serum
albumin antibodies (anti-BSA) 37,
antibodies reacting with ICA69 38-40, anti-insulin receptor
antibodies 41,
antibodies to heat shock proteins 42-44, anti-topoisomerase II
45 anti-ganglioside 46,
lysophospholipids 47and
GLIMA38 a membrane glycoprotein 48and antibodies to a series of
autoantigens identified by screening islet libraries such as ICA12 49, 50.
Finally, a subset of autoantibodies termed anti-islet cell surface antibodies
are currently rarely measured as most recent studies have failed to demonstrate
disease specificity 51.Recently described autoantigens include
osteopontin 52,
importin 53,
antibodies reacting with “peri-islet Schwann cells/ GFAP/S100beta (glial
fibrillary acidic protein)” surrounding islets 54,
densin and filtrin55 and antibodies
reacting with CD38 56.There are almost certainly additional
specificities awaiting discovery.

Table 10.1: Subset of
Biochemically Characterized Autoantigens

Sensitivity

Comment

Insulin

49-92%

Higher sensitivity young children/rapid progressors

GAD (Glutamic acid Decarboxylase)

65-75%

Higher sensitivity adult onset type 1A

ZnT8

65-75%

Islet Zinc Transporter

ICA512/IA-2

74%

Tyrosine Phosphatase like molecule

IA-2β/Phogrin

61%

Tyrosine Phosphatase like molecule

Carboxypeptidase H

10%

Infrequent

GLIMA38

19%

amphiphilic membrane glycoprotein

Despite the importance of autoantibodies for disease
prediction 28,
it is likely that anti-islet autoantibodies do not by themselves cause the beta
cell destruction that leads to Type 1A diabetes.Nevertheless B lymphocytes are important for
the development of Type 1 diabetes and results of the TrialNet studies of anti-B
cell antibodies (anti-CD20) in new onset diabetes demonstrated slowing of loss
of C-peptide.In the NOD mouse,
anti-CD20 antibody treatment decreases development of diabetes 57.
In addition, Naji and coworkers have demonstrated the importance of
transplacental autoantibodies for progression to diabetes of NOD mice 58.

The most cogent evidence of lack of direct damage by
autoantibodies is the lack of diabetes in infants born to antibody positive
mothers, or women who developed Type 1A diabetes during pregnancy.
Autoantibodies (ICA,
anti-insulin, and anti-GAD autoantibodies) are readily detected in the serum of
infants of such antibody-positive mothers in the absence of diabetes in the
child. It will take careful epidemiological studies to determine if specific
autoantibodies (e.g. spontaneous and not insulin therapy induced insulin
antibodies) predispose children to develop type 1 diabetes. A report by Ziegler
and coworkers indicates that the presence of anti-islet autoantibodies at birth
(GAD65 and IA-2 autoantibodies, but not insulin autoantibodies) of offspring of
mothers with type 1A diabetes was associated with a decrease 59 in the development of
anti-islet autoantibodies for children followed in the BabyDiab study 60. The five year risk of
developing anti-islet autoantibodies and diabetes was 1.3% and 1.1% for
offspring with newborn anti-islet autoantibodies versus 5.3% and 3%
respectively of newborns who were negative for anti-islet autoantibodies. Of
note, a child with genetic B cell deficiency and no antibodies progressed to
type 1 diabetes 61.
On the other hand studies in the NOD mouse indicate that both B lymphocytes and
transplacental autoantibodies (insulin autoantibodies detected) greatly
increase the development of diabetes 57-59, 61-63.As illustrated in the Figure 10.2 below, if
the mother NOD mouse was unable to make autoantibodies, even if
"antibodies" (e.g. transgenic anti-Hen Egg Lysozyme antibody gene)
could be produced, the development of diabetes in offspring was greatly
decreased.

Figure 10.2
Development of diabetes is greatly reduced if maternal autoantibodies are not
present during pregnancy, presumably due to a lack of transplacental
autoantibodies. HEL=Transgenic producing antibodies to Hen Egg Lysozyme; KO=IgM
knockout; DBA/2 strain of mouse used as foster mother; SCID=Severe Combined
Immunodeficient Mother

General Assay
Methodology

There are multiple assay formats for the detection of
disease associated autoantibodies, and similar to many fields of autoimmunity,
the first useful assay for islet autoantibodies utilized indirect immunofluorescence
with frozen sections of human pancreas as substrate (the ICA assay-see below) 1.The other major format for determination of
islet autoantibodies consists of variations on the theme of fluid phase assays
including recently described luciferase immunoprecipitation assays 19
and electrochemiluminescence assays where the autoantibodies couple a
biotinylated autoantigen and fluorophore labeled autoantigen20.
In fact the methodology used for the discovery of the radioassay by Berson and
Yalow, utilizing insulin antibodies produced by patients treated with insulin,
was the basis for the discovery of insulin autoantibodies 2, 64. For most autoimmune
disorders and for basic immunologic research, ELISA assays employing plate
bound antigen are the standard. Multiple workshops utilizing sera from patients
with type 1 diabetes and multiple mouse strains have demonstrated that standard
ELISA formats lack both sensitivity and specificity compared to fluid phase
radioassay formats. For insulin autoantibodies, the ELISA formats were able to
detect high capacity antibodies following insulin immunization, but not the
insulin autoantibodies of prediabetic individuals 10.
This inability of human insulin autoantibodies to bind to plate bound insulin
is specific to human autoantibodies as the insulin autoantibodies of NOD mice
can readily be detected in a plate bound ELISA format and we have described a
highly sensitive and specific assay for such autoantibodies, equivalent to
radioassays 65.We believe insulin bound to plastic plates
obscures the limited key determinant(s) recognized by human anti-insulin
autoantibodies.

In addition when one analyzes thousands of individuals with
ELISA formats there is always a subset of sera that react with the wells of the
plate itself or unique epitopes created by plate bound proteins. A small
percentage of such sera (e.g. 1%) in studies such as DAISY (Diabetes
Autoimmunity Study of the Young) where individuals are sampled on multiple
occasions over time, would result in enough false positives to invalidate
efforts to discover environmental, immunogenetic, and diabetes predictive
parameters 22, 66. It is possible to
develop modified ELISA like assays that utilize fluid phase reactivity of
antigen and antibody(see discussion of method below). In international workshops an assay for GAD65
autoantibodies (company RSR developed assay and distributed by Kronus) has
attained a level of specificity and sensitivity equivalent to the radioassays,
though in the most recent 2009 DASP workshop multiple laboratories using this
modified ELISA had excellent sensitivity but only 95% specificity (Mueller oral
communication).For many applications,
5% false positives would be problematic.

A disadvantage of the current fluid phase radioassays is
that they utilize low levels of radiation (usually 125I, 35S
-methionine, and 3H-leucine)(Figure 10.3). They are however often
performed in a format in 96-well format that is as convenient as the ELISA
format 67-69. It is likely that
many of the clinical assays (but not all, e.g. transglutaminase) that utilize
ELISA formats for other autoimmune disorders are utilizing assays with
compromised specificity and sensitivity, and the assay format development has
just not been optimized and directly compared with modern fluid phase
radioassays (e.g. Farr assay versus, protein A based assay, versus ELISA assay
for anti-DNA autoantibodies) 70.

Most investigators assay GAD65 and IA-2 anti-islet
autoantibodies in a high throughput 96-well format(Figure 10.3)where labeled antigen is
incubated with patient sera, and then both are placed in 96-well filtration
plates, where a "bead" (e.g. sepharose) with coupled protein A and or
protein G is added, and bound from free radioactivity is separated by
filtration washing, and then scintillation fluid is added directly to the 96-well
filtration plates, and counting is performed on multichannel beta counters able
to handle the plates 58.
Even the assay for insulin autoantibodies utilizing 125Iinsulin is performed in the same manner,
detecting with beta counting emission from 125I(Figure 10.3). A major advance was the simple
production of labeled autoantigen by in vitro transcription and translation of
cDNAs to produce the label for the fluid phase radioassays. For example, we
have utilized a combined GAD65 and ICA512bdc radioassay in which GAD65 is
labeled with 3H-leucine and ICA512(IA-2) is labeled with 35S
-methionine. It has been our experience in setting up multiple
such autoantibody assays that approximately 2/3 of the time an assay using such
in vitro labeled autoantigen works, and if the assay does not work on the first
try, it is unlikely to be modified to work. At present we routinely determine
GAD65, ICA512bdc, IA-2ic, IA-2 full length, ZnT8, 21-hydroxylase and
transglutaminase autoantibodies with this methodology 71-75. Such assays are not
useful if post-translational modifications, or particular folding of the
protein is essential that is not reproduced in the in vitro production of the
antigen. With this in vitro translation and transcription methodology minimal
protein preparation is needed and following the kit generation of the labeled
product we simply perform size separation to produce the labeled antigen.Two harmonized NIDDK assay have been created
(for GAD65 and IA-2 autoantibodies) which depended in part on using the same
standard sera as well as identical reagents. 76

There are a number of modifications proposed and implemented
for the determination of defined islet autoantibodies that can detect
autoantibodies with various degrees of sensitivity and specificity relative to
the best standard fluid phase radioassays 77-80. In particular the
GAD65 autoantibody described by Smith and coworkers 79utilizes a novel ELISA format in which a low concentration of the
GAD antigen on the plate captures the autoantibody, and then biotinylated GAD
in the fluid phase is added and is captured by the second binding site of the
autoantibody, and it is the biotinylated GAD65 that is detected to produce the
non-isotopic signal. This assay performed well in the previous Immunology of
Diabetes/CDC DASP workshop, but a similar format for IA-2 autoantibodies did
not have an equivalent sensitivity to the standard fluid phase radioassays(oral
discussion at IDS-Cambridge and in current 2009 DASP specificity for multiple
ELISA assays was only approximately 95%).There is also now a similar ZnT8 commercial assay kit.Human insulin autoantibodies can be measured
with an electrochemiluminescent (ECL) insulin/proinsulin assay20.A reported ECL insulin autoantibody assay
detected antibodies of NOD mice despite binding of insulin to plate81.I believe it is unlikely for this assay to
detect prediabetic insulin autoantibodies in that even though NOD insulin
autoantibodies react with plate bound insulin, prediabetic IAA do not 10.There is a need for "point of
service" anti-islet autoantibody assays as at present a major portion of
the expense of screening for anti-islet autoantibodies relates to handling of
the serum specimen and communication with families.We have initial experience with
electrochemiluminescent assays for insulin, GAD6S and IA-2 autoantibodies that
utilize plate capture and ruthenium labeling of autoantigen which has potential
for development of non-radioactive assays